Phytochemical Analysis and Profiling of Antioxidants and Anticancer Compounds from Tephrosia purpurea (L.) subsp. apollinea Family Fabaceae

Tephorosia purpurea subsp. apollinea was extracted with methanol and n-hexane to obtain sub-fractions. The chemical compounds identified with GC-MS and HPLC in T. purpurea subsp. apollinea extracts showed antioxidant and anticancer properties. The antioxidant and anticancer activities were investigated using DDPH and ABTS assays, and MTT assay, respectively. Stigmasta-5,24(28)-dien-3-ol, (3 β,24Z)-, 9,12,15-octadecatrienoic acid methyl ester, phytol, chlorogenic acid, and quercetin were the major chemical compounds detected in T. purpurea subsp. apollinea. These compounds possessed antioxidant and anticancer properties. The methanol extract showed antioxidant properties with DDPH and ABTS radical scavenging of 84% and 94%, respectively, relative to ascorbic acid and trolox. The anticancer effects of T. purpurea subsp. apollinea against the cancer cell lines MCF7 (IC50 = 102.8 ± 0.6 μg/mL), MG63 (IC50 = 118.3 ± 2.5 μg/mL), T47D (IC50 = 114.7 ± 1.0 μg/mL), HeLa (IC50 = 196.3 ± 2.3 μg/mL), and PC3 (IC50 = 117.7 ± 1.1 μg/mL) were greater than its anticancer effects against U379 (IC50 = 248.4 ± 7.5 μg/mL). However, it had no adverse effects on the normal cells (WI38) (IC50 = 242.9 ± 1.8 μg/mL). Therefore, the major active constituents presented in T. purpurea subsp. apollinea can be isolated and studied for their potential antioxidant and anticancer effects against breast, cervical, and prostate cancers and osteosarcoma.


Introduction
Despite the advancements in different therapeutic strategies, cancer continues to be a foremost cause of mortality worldwide. Conventional cancer therapies include radiotherapy, chemotherapy, and surgery. However, serious side effects and drug resistance to these treatments decrease their effectiveness [1]. The development of such problems, particularly chemotherapy drug resistance, is considered one of the main contributors to cancer-related mortality. Different multiple mechanisms are employed by cancer cells to decrease the therapeutic benefits of anticancer drugs. These include, but are not limited to, alteration in expressions of targeted proteins, DNA repair mechanisms, drug detoxification, and inactivation of medication by several catalytic proteins [2][3][4]. Therefore, there is a need to identify novel drug targets, as well as use of unique sources of anticancer drugs such as natural products [5,6]. As anticancer agents, natural products constitute an acceptable therapeutic strategy due to their availability, applicability, and low cytotoxicity. Importantly, they may provide new therapeutic strategies for combating drug resistance seen with traditional chemotherapy via several mechanisms of action.
Numerous secondary metabolites with different chemical structural diversities and biological effects have demonstrated significant potential in the treatment of various carcinomas [7]. Overall, natural products have the potential to be promising sources of novel anticancer drugs, making them an essential field of research.
The imbalance between the generation of oxidants and the antioxidant defense mechanism in the human body is known as oxidative stress [8]. Oxidative stress may cause cardiovascular and respiratory diseases such as coronary heart disease, hypertension, and chronic obstructive pulmonary disease [8]. It also may produce neurodegeneration-associated ailments such as Alzheimer's and Parkinson's diseases [8]. Additionally, oxidative stress strongly contributes to cancer development and metastasis [8]. Oxygen-and nitrogenderived free radicals are reactive entities that are involved in the induction of oxidative stress that causes cellular damage [9]. Antioxidants from natural products have been widely investigated for preventing oxidative stress [10] and for the treatment of different illnesses, e.g., inflammation, carcinoma, diabetes, and neurodegenerative diseases [11]. For example, it has been demonstrated that Portulaca oleracea extract produced potential protective effects against neuroinflammatory disease, memory loss, and oxidative stress induced by lipopolysaccharide in mice [12].
Tephrosia belongs to the family Fabaceae [13]. It is widespread in tropical and subtropical regions of the world [14]. It has been widely used in folk medicine [14]. Tephrosia purpurea (L.) is a member of genus Tephrosia [13]. It has two sub-species (subsp.), leptostachya (DC.) Brummitt and apollinea (Delile) Hosni and El-Karemy [13]. Both sub-species grow in Egypt [13]. The T. purpurea subsp. apollinea is distributed in the Nile Delta, Nile banks, and desert wadis, especially Wadi Allaqi (Nubia), Gebel Elba (southern Egypt), and Sudan [13]. Several studies have revealed that the flavonoids and phenolic components of the genus Tephrosia have potent pharmacological effects, including pesticidal, insecticidal, and anticancer properties, especially against the human breast cancer cell line MCF7 [15]. For example, rotenoids, terpenoids, sterols, essential oils, and fixed oils have been identified as chemical compounds in the Tephrosia species [16]. Moreover, the Tephrosia species contains a large amount of flavonoids with antioxidant and anticancer effects [16]. The objective of this study is to analyze the chemical compounds in solvent subfractions of T. purpurea subsp. apollinea, in addition to investigation of their antioxidant and anticancer effects, for the first time.
A curve was plotted showing the percentage of DPPH and ABTS radical scavenging activities vs. concentration, and the concentration of the sample required to scavenge 50% of DPPH and ABTS free radicals, which is known as IC 50 , was determined using GraphPad Prism 8 ( Figure 6). The result of the DPPH assay showed that the methanol extract of T. purpurea subsp. apollinea had IC 50 of 46.7 ± 0.7 µg/mL. However, ascorbic acid (a positive control) had IC 50 of 4.8 ± 0.1 µg/mL. At a concentration of 1000 µg/mL of T. purpurea subsp. apollinea methanolic extract, the DPPH scavenging percentage was 84.17% (Table 4). Additionally, data from ABTS studies were comparable to the results of DPPH radical assay, and the antioxidant effect of T. purpurea subsp. apollinea methanolic extract had IC 50 of 46.7 ± 2.6 µg/mL. However, trolox (a positive control) had IC 50 of 2.9 ± 0.1 µg/mL. At a concentration of 150 µg/mL of T. purpurea subsp. apollinea methanolic extract, the ABTS scavenging percentage was 94.56% (Table 5). This may indicate potential antioxidant properties of T. purpurea subsp. apollinea.
The identification of the compounds of n-hexane subfraction extract of T. purpurea subsp. apollinea was performed with GC-MS. The majority of compounds in n-hexane subfraction extract possess antioxidant and anticancer properties. The identified phenol with antioxidant activity was 2,4-di-tert-butylphenol [37]. Additionally, hexadecanoic acid methyl ester [39], 9,12,15-octadecatrienoic acid methyl ester, which was present at the highest level relative to the other constituents [42], and methyl stearate [45] were the identified fatty acids methyl esters that are known to exert antioxidant and anticancer effects. However, the fatty acid methyl ester cis-5,8,11-eicosatrienoic acid methyl ester exerts anti-inflammation-related effects [48]. The identified diterpene was phytol, of which there was a considerable amount relative to the other components, and it has antioxidative stress and anticancer activities [43,44]. The long-chain alcohol 1-heptacosanol has been reported to be an antioxidant [46]. The identified fatty acid amide with antioxidant properties was palmitoleamide [50]. Bis(2-ethylhexyl) phthalate exerts antioxidant and antitumor effects [52]. Additionally, gamma-sitosterol is a steroid that has been reported to possess anticancer activity [54]. The other identified compounds have not been evaluated for either antioxidant or anticancer properties; however, they have been evaluated for different biological activities. For example, pentadecane [38], tetradecane [36], and 2-methyltetracosane [49] are alkanes with antibacterial effects. An aliphatic hydrocarbon (heneicosane) [41] and carbonyl compound (tributyl acetylcitrate) [47] have been reported to possess pesticidal and antimicrobial activities, respectively, while acetic acid, 10,11dihydroxy-3,7,11-trimethyl-dodeca-2,6-dienyl ester [51] and pentafluoropropionic acid octadecyl ester [40] are the identified esters and have been reported with insecticidal and antimicrobial properties, respectively.
The phenolic acids and flavonoids found in methanolic T. purpurea subsp. apollinea extract may have the potential to actively participate in the antioxidant and anticancer effects on the tested carcinoma cells. These phytochemical components were identified with the HPLC apparatus, which analysis showed the presence of phenolic acid (gallic acid), which has been reported to have antioxidant and anticancer activities [55,56]. Another phenolic acid identified in T. purpurea subsp. apollinea methanolic extract was ferulic acid, which has also been reported to have antioxidant and anticancer activities [65]. Chlorogenic acid [57], pyrocatechol [60], and coumaric acid [63] were the identified phenolic compounds in T. purpurea subsp. apollinea extract with antioxidant and anticancer properties. The identified gallate ester in the extract (methyl gallate) has also been reported to have antioxidant and anticancer activities [58]. Additionally, caffeic acid was the identified polyphenol, and it is known to exert antioxidative stress and anticancer activities [59]. The tannin (ellagic acid) also has antioxidant and anticancer potential [62]. The flavonoids rutin [61] and quercetin [68] have been reported to have antioxidant and anticancer properties. Additionally, naringenin [66] and hesperetin [72] were the identified flavanones known to have antioxidant and anticancer properties. These findings agree with the results reported in the literature where the flavanones (-)-pseudosemiglabrin isolated from T. apollinea, which has a close affiliation to T. purpurea subsp. apollinea showed anticancer effects against leukemia, breast, and prostate cancers [73]. Daidzein is an isoflavone that has also been reported to exert antioxidant and anticancer properties [67]. Cinnamic acid is monocarboxylic acid present in T. purpurea subsp. apollinea, and studies have shown that it has antioxidant and anticancer properties [69]. The phenolic aldehyde found in the extract with reported antioxidant and anticancer properties was vanillin [64]. Apigenin [70] and kaempferol [71] were the identified flavone and flavonol, respectively, and have been reported to have antioxidant and anticancer properties. These results agree with existing data on different extracts from T. apollina, which contained flavones (semiglabrin 1, pseudosemiglabrin 2, glabratephrin 3, and apollinine 4) [74]. Interestingly, these extracts showed various degrees of antioxidant and anticancer effects against hepatocellular carcinoma (HepG2), colorectal carcinoma (HC116), and prostate cancer (PC3) [74]. Additionally, another prenylated flavone (isoglabratephrin) isolated from T. apollinea showed anticancer activities against prostate cancer (PC3) and pancreatic cancer (PANC1) through induction of chromatin disruption and nuclear damage [75]. This is consistent with the anticancer effects of T. purpurea subsp. apollinea against breast cancer (MCF7), ductal breast cancer (T47D), osteosarcoma (MG63), cervical cancer (HeLa), and prostate cancer, as seen in this research. However, the anticancer effect was weak against leukemia, when compared with other cancer cell lines. Therefore, T. purpurea subsp. apollinea exerted anticancer impact on all cells investigated, except leukemia. However, the anticancer effects of T. purpurea subsp. apollinea were without any selective cytotoxicity to cancer cells, relative to non-cancer cell lines (WI38). In this study, T. purpurea subsp. apollinea methanolic extract also demonstrated potential dose-dependent antioxidant activities, when compared to ascorbic acid. This result is consistent with the findings of dose-dependent inhibition of DPPH radical by T. apollinea methanolic extract [76]. Additionally, different extracts of T. apollinea have been reported to have antioxidant activities when assessed for radical scavenging, TAC, anti-lipid peroxidation, and GSH level [77]. Therefore, T. purpurea subsp. apollinea may have potential antioxidant properties due to the various identified antioxidant compounds, and it may also have anticancer properties due to the presence of different anticancer compounds.

Plant Material
The aerial parts of T. purpurea subsp. apollinea were collected from the Al-Mansoura city (31 • 02 27.2 N 31 • 22 42.6 E), Delta region, Egypt, at the flowering stage in March 2022, and the voucher sample was kept at its herbarium (CAIH-21 /23-5) in Cairo, Egypt after it was authenticated by a plant taxonomist Professor Iman Hussein Salama Al-Gohary. Following rinsing in running water and shade dehydration for 1 week and 3 days at 25 • C, the specimen was ground to powder [7].

Preparation of Methanolic Extract and GC-MS Studies
Utilizing the cold percolation method, 200 g of the above sample of the plant was subjected to extraction. Subsequently, the extract was exposed to three separate applications of 500 mL of 70% methanol for 72 h at 25 • C. The methanol extract was filtered using a Buchner funnel. Then, the remaining methanol was entirely removed from the methanol extract using a rotary evaporator and concentration at low pressure at 40 • C. The sediment was dried in a desiccator to produce a dry weight yield of 20.68 g/100 g of T. purpurea subsp. apollinea, and GC-MS analysis was employed to identify the bioactive components [7].
A TRACE 1310 gas chromatograph connected to an ISQLT MS single quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used. The data were obtained from the GC-MS at 70 eV ionization voltage, EI ionization mode, DB5-MS column with an internal diameter of 0.25 mm (J & W Scientific, Folsom, CA, USA), and the temperature was programmed in this manner: 3 min at 40 • C, 5 min at 280 • C, 1 min at 290 • C and constant at 7.5 • C/min. The detector and injector temperatures were adjusted to be 300 and 200 • C, respectively. The flow rate of the carrying gas (helium) was constant at 1 mL/min. The WILEY and NIST Mass Spectral Data Base was used as a search library [7].

Sub-Fractionation Using n-Hexane and GC-MS Studies
In 250 mL of distilled water, 2.5 g of lyophilized crude methanolic extract of T. purpurea subsp. apollinea was re-dissolved. Then, for 24 h, the re-dissolved crude extract was separated with n-hexane using a separatory funnel, followed by the use of a rotating evaporator at a temperature of 40 • C and lower pressures to obtain the n-hexane layer in a dry form. A GC-MS analysis was performed on the dried portion (1 mg) [78,79].
A split-splitless injector was used in tandem with the Shimadzu GCMS-QP2010 (Shimadzu, Tokyo, Japan). The mass spectra were acquired using a Restek 30-m Rtx-5MS column with an internal diameter of 0.25 mm (Chrom Tech, Bellefonte, PA, USA). The starting temperature of the column was raised to 300 • C at a rate of 5 • C/min for 5 min and was then held constant there for 2 min (isothermal). The injector temperature was 250 • C. Helium carrier gas was at a flow rate of 1.41 mL per min. The ion source of 200 • C, ionization voltage of 70 eV, and filament emission current of 60 mA were applied to acquire all mass spectra. The sample (1% v/v) dilution injection was performed via a split mode [78].

Quantification of Phenolics and Flavonoids
Phenolics and flavonoids were quantified using the standard Folin-Ciocalteu technique [79,80]. The optical density of the bluish reaction solution was read at a maximum λ max of 725 nm after one hour using the Unicam UV-visible Spectrometer, with distilled H 2 O as blank. A calibration curve of gallic acid was plotted. The results were calculated in terms of milligrams of gallic acid equivalents (GAE) per gram dry weight [79,80].
The total flavonoid content was determined using an aluminum chloride colorimetric method [79,80]. The extract was diluted 1:6 (v:v) with distilled water, and the mixture was then added to 75 µL of NaNO 2 (5%), followed by the addition of 10% AlCl 3 .6H 2 O (150 µL) after 6 min to the mixture and it was let to stand for an additional 5 min. After adding 1 M NaOH solution (0.5 mL), 2.5 mL of distilled water was added to the mixture. Optical density was recorded at 510 nm against a blank of distilled water. A standard calibration curve was developed using (+)-catechin. The findings were calculated as mg of catechin equivalents (CE) for each gram [79,80].

Standards
The phenolic compounds were investigated with HPLC reagents (acetonitrile, methanol, and trifluoroacetic acid) purchased from SDS (Peypin, France). The distilled water was obtained from Milli-Q (Millipore, MA, USA). All standards used (methyl gallate, coffeic acid, etc.) were provided by Sigma (St. Louis, MO, USA) and had a 98% level of purity [79,80].

HPLC Quantitation of Phenolics
The MeOH extract of T. purpurea subsp. apollinea (0.20 g) was solubilized in 2 mL of acetonitrile. The identification of phenolic compounds was performed using an Agilent 1260 HPLC instrument (Agilent Technologies, Santa Clara, CA, USA). An Eclipse C18 column (4.6 mm × 250 mm i.d., 5 µm) was used to separate the phenolic compounds, with a mobile phase comprised of H 2 O (A) and CF 3 COOH (0.05%) in acetonitrile (B) at a flow rate of 0.9 mL/60 s.
A linear gradient was applied to configure the mobile phase. Sample monitoring was performed at 280 nm using a multiple λ max detector. For each sample solution, 5 µL was injected into the column at 40 • C. Standards were prepared as stock solutions of 10 mg/50 mL in methanol. Then, the standards were loaded into HPLC after being diluted. The flavonoids and phenolic acids in the methanolic extract from T. purpurea subsp. apollinea were identified and quantified using Equation (1), and the results were calculated in terms of mg/100 g dry weight [79,80]. The 2,2-diphenylpicrylhydrazyl (DPPH) neutralizing potential of methanol extract of T. purpurea subsp. apollinea was investigated. The methanolic extract of T. purpurea subsp. apollinea was serially diluted to various concentrations, i.e., 1.95, 3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 µg/mL. Each methanolic extract concentration (1 mL) was added to 3 mL of 0.1 mM DPPH solubilized in methanol, followed by shaking and placing it into a dark chamber for 30 min. When DPPH reacts with an H-donating antioxidant, it is scavenged, thereby resulting in a decrease in absorbance [80]. The optical density of each concentration was measured at 517 nm in a UV-Vis spectrophotometer. Ascorbic acid was used as an antioxidant standard. All the values were measured in triplicate.

ABTS Antioxidant Assay
Distilled water was used to bring up a 50-mL volumetric flask to mark after dissolving 192 mg of 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in a small volume of water and transferring it to the flask, followed by addition of 1 mL of this solution to 17 µL 0.14 M K 2 S 2 O 8 , and incubation in darkness for 24 h. To prepare the final ABTS dilution for the T. purpurea subsp. apollinea extract, 1 mL of reaction mixture was added to 49 mL of methanol. Then, 0.190 mL of newly made ABTS solution was mixed with 0.010 mL of plant extract in a 96-well plate and kept in the dark for 30 min. Thereafter, a decrease in ABTS OD was read at 734 nm in a FluoStar Omega microplate reader. Trolox was used as an antioxidant standard. All the values were measured in triplicate.

Measurement of IC50
The IC50 values of DPPH and ABTS antioxidant assays for samples and controls were calculated using GraphPad Prism 7. The IC 50 values were calculated as shown in Equation (2):

Determination of Anticancer Effect
Breast cancer cell lines (MCF7), osteosarcoma (MG63), breast ductal carcinoma (T47D), leukemia (U937), as well as HeLa, PC3, and healthy pulmonary fibroblast were supplied by the tissue culture laboratory at Vacsera, Egypt. The culturing procedure was maintained sterile, utilizing a laminar airflow cabinet. The cells were cultured in Roswell Park Memorial Institute medium (RPMI 1640). The medium was provided with antibiotics (streptomycin and penicillin) as well as antifungal agents (amphotericin B) and L-glutamine. It was also supplemented with 10% heat-inactivated fetal bovine serum [80].

Viability Evaluation
Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide (MTT) assay. The principle of the MTT assay is that through mitochondrial reduction, purple-colored crystals are produced from the yellowish MTT [80]. A 96-well microplate was used for inoculation, and 100 µL of the Roswell Park Memorial Institute medium (RPMI 1640) was added to each well. A fully formed monolayer sheet was produced by incubation of the microplate for 24 h at 5% CO 2 , 37 • C, and 95% humidity. When the cells had formed a confluent layer, the growth medium was discarded.
Using a growth medium, serial dilutions of the dimethyl sulfoxide (DMSO)-solubilized extract were produced at concentrations of 31.25, 62.5, 125, 250, 500, and 1000 µg/mL [80]. Using a multichannel pipette, the cells were transferred to 0.10 mL of each extract concentration in triplicate before being dispersed in 96-well plates, followed by incubation of the extract-treated cells for 24 h at 37 • C and 5% CO 2 . Control cells were incubated without the addition of the stem and leaf extracts. Thereafter, 20 µL of MTT solution (5 mg/mL) in PBS was added to each well, followed by mixing, which was performed for 5 min at 150 rpm. After that, incubation was maintained for 4 h. Then, the formazan crystals were taken up in 200 µL of DMSO and vigorously agitated. A microplate reader was used to measure the optical density of the formazan solution at 560 nm, with values corrected using a background reference λ of 620 nm [80]. Each experiment was performed three times.

Measurement of IC 50
The IC 50 profiles of methanol extract of T. purpurea subsp. apollinea and a positive control for cancer and healthy cell lines were determined with GraphPad Prism version 7 (GraphPad Software Inc., San Diego, CA, USA). The IC 50 values were computed as shown in Equation (3) [80]. The data obtained were subjected to non-linear regression to obtain 50% effective concentrations (EC50) and cytotoxic concentration (CC50), with 95% confidence intervals.

Criteria for Selectivity
The selectivity index (SI) is IC 50 for a healthy cell (WI38) divided by IC 50 for a cancerous cell. SI values less than 3 indicate non-specificity to cancer cells [81]. The SI values of methanolic extract of T. purpurea subsp. apollinea were calculated using Equation ( Morphologies of cells treated with the various methanol T. purpurea subsp. apollinea extract concentrations were investigated under light microscopy at 10× objective lens, total magnification = 100×.